ABSTRACT
Chimeras of the well-characterized minimal hammerhead 16 and nine extended hammerheads derived from natural viroids and satellite RNAs were constructed with the goal of assessing whether their very different peripheral tertiary interactions modulate their catalytic properties. For each chimera, three different assays were used to determine the rate of cleavage and the fraction of full-length hammerhead at equilibrium and thereby deduce the elemental cleavage ( k 2) and ligation ( k -2) rate constants. The nine chimeras were all more active than minimal hammerheads and exhibited a very broad range of catalytic properties, with values of k 2 varying by 750-fold and k -2 by 100-fold. At least two of the hammerheads exhibited an altered dependence of k obs on magnesium concentration. Since much less catalytic diversity is observed among minimal hammerheads that lack the tertiary interactions, a possible role for the different tertiary interaction is to modulate the hammerhead cleavage properties in viroids. For example, differing hammerhead cleavage and ligation rates could affect the steady state concentrations of linear, circular, and polymeric genomes in infected cells.
Subject(s)
RNA, Catalytic/metabolism , Base Sequence , Biological Assay , Catalysis/drug effects , Magnesium/pharmacology , Molecular Sequence Data , Nucleic Acid Conformation/drug effects , RNA, Viral/chemistry , RNA, Viral/genetics , RNA, Viral/metabolismABSTRACT
Two structurally homologous guanosine triphosphatase (GTPase) domains interact directly during signal recognition particle (SRP)-mediated cotranslational targeting of proteins to the membrane. The 2.05 angstrom structure of a complex of the NG GTPase domains of Ffh and FtsY reveals a remarkably symmetric heterodimer sequestering a composite active site that contains two bound nucleotides. The structure explains the coordinate activation of the two GTPases. Conformational changes coupled to formation of their extensive interface may function allosterically to signal formation of the targeting complex to the signal-sequence binding site and the translocon. We propose that the complex represents a molecular "latch" and that its disengagement is regulated by completion of assembly of the GTPase active site.
Subject(s)
Bacterial Proteins/chemistry , Guanosine Triphosphate/analogs & derivatives , Heterotrimeric GTP-Binding Proteins/chemistry , Receptors, Cytoplasmic and Nuclear/chemistry , Signal Recognition Particle/chemistry , Thermus/chemistry , Amino Acid Motifs , Bacterial Proteins/metabolism , Binding Sites , Catalysis , Crystallography, X-Ray , Dimerization , Guanosine Triphosphate/metabolism , Heterotrimeric GTP-Binding Proteins/metabolism , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Subunits , Receptors, Cytoplasmic and Nuclear/metabolism , Signal Recognition Particle/metabolismABSTRACT
The GTPases Ffh and FtsY are components of the prokaryotic signal recognition particle protein-targeting pathway. The two proteins interact in a GTP-dependent manner, forming a complex that can be stabilized by use of the non-hydrolyzable GTP analog GMPPCP. Crystals of the complex of the NG GTPase domains of the two proteins have been obtained from ammonium sulfate solutions. Crystals grow with several different morphologies, predominately as poorly diffracting plates and needle clusters, but occasionally as well diffracting rods. It has been demonstrated that all forms of the crystals observed contain an intact complex. Diffraction data to 2.0 A resolution have been measured.
Subject(s)
Bacterial Proteins/chemistry , Escherichia coli Proteins/chemistry , GTP Phosphohydrolases/chemistry , Guanosine Triphosphate/analogs & derivatives , Guanosine Triphosphate/chemistry , Receptors, Cytoplasmic and Nuclear/chemistry , Signal Recognition Particle/chemistry , Thermus/enzymology , Crystallization , Crystallography, X-Ray , Protein Structure, TertiaryABSTRACT
The structural basis for the GTP-dependent co-translational targeting complex between the signal recognition particle (SRP) and its receptor is unknown. The complex has been shown to have unusual kinetics of formation, and association in vivo is likely to be dependent on catalysis by the SRP RNA. We have determined conditions for RNA-independent association of the 'NG' GTPase domains of the prokaryotic homologs of the SRP components, Ffh and FtsY, from Thermus aquaticus. Consistent with previous studies of the Escherichia coli proteins, the kinetics of association and dissociation are slow. The T. aquaticus FtsY is sensitive to an endogenous proteolytic activity that cleaves at two sites--the first in a lengthy linker peptide that spans the interface between the N and G domains, and the second near the N-terminus of the N domain of FtsY. Remarkably, this second cleavage occurs only on formation of the Ffh/FtsY complex. The change in protease sensitivity of this region, which is relatively unstructured in the FtsY but not in the Ffh NG domain, implies that it undergoes conformational change on formation of the complex between the two proteins. The N domain, therefore, participates in the interactions that mediate the GTP-dependent formation of the targeting complex.
